Although accumulating evidence suggests that p53-mediated cell cycle arrest, senescence and apoptosis are not absolutely required for its tumor suppression, it remains unclear how p53 executes the tumor suppression activity, in the absence of its canonical functions. In our preliminary study, we found that SLC7A11, a component of the cystine/glutamate antiporter, is a transcriptional target of p53. By suppressing SLC7A11 expression, p53 sensitizes cells to ferroptosis, an iron-dependent non-apoptotic form of cell death. Notably, p533KR fully retains the ability to regulate SLC7A11 expression and ferroptosis. Analysis of mutant mice shows that p53- mediated ferroptosis contributes significantly to the embryonic lethality associated with loss of Mdm2. Moreover, SLC7A11 is overexpressed in human tumors, and p53-mediated regulation of SLC7A11 and ferroptosis modulates cancer cell survival. The central hypothesis to be tested here is whether p53-mediated effects on SLC7A11 expression and ferroptosis act as an independent mechanism in tumor suppression, particularly when the p53-mediated pathways of cell-cycle arrest, senescence and apoptosis are inoperative. The proposed studies include the following two specific aims. In Aim 1, we will dissect the mechanisms of p53-mediated regulation of SLC7A11 in cancer metabolism and cancer cell survival. To investigate the precise role of p53 in modulating cancer metabolic pathways through suppressing SLC7A11 expression in vivo, we will further examine the roles of both p53-wt and p533KR in regulating cellular uptake of cystine as well as other metabolic changes. Moreover, we will dissect the potential dysregulation of the p53/SLC7A11/ferroptosis pathway by tumor-derived point mutants. We will investigate whether p53-mediated ferroptosis is regulated in both normal and cancer cells under different types of stressed and unstressed conditions. Finally, to elucidate the mechanism of p53-mediated SLC7A11 expression, we will identify cofactors and signaling that are critical for SLC7A11 expression in vivo. In Aim 2, we will evaluate the biological significance of p53-mediated regulation of SLC7A11 in vivo. We will first examine whether mdm2 inhibition can promote the ability of p53 in regulating SLC7A11 expression and activating ferroptosis, which may improve the efficacy of nutlin-3 in inducing cancer cell death. Moreover, since SLC7A11 overexpression is observed in several forms of human cancer, it is very likely that p53-mediated repression contributes significantly to its mediated tumor suppression. We will examine whether SLC7A11 status modulates the effect of p53 on Myc-induced tumorigenicity using the E-Myc/ p53 lymphoma model.